Abstract Mycobacterium tuberculosis (Mtb) is a human pathogen that causes significant morbidity and mortality worldwide. An increasing number of tuberculosis cases are caused by multi-drug and extreme-drug resistant Mtb strains underscoring the need for novel therapeutics. The efficient host response to Mtb infection depends on the production of IL-1 which is mediated by the inflammasome complex in macrophages and dendritic cells. The Absent In Melanoma 2 (AIM2) protein is one of the inflammasome components and it is able to bind to double stranded DNA of pathogens such as Francisella and Listeria in the cell cytosol and then induces assembly and activation of the inflammasome complex. Here we report the novel and surprising finding that Mtb is able to inhibit the activation of the host cell AIM2-inflammasome. We hypothesize that genetic screens will be able to identify the Mtb gene(s) mediating this novel immune evasion strategy. We propose to use a Gain-of-Function (GoF) genetic screen by comparing the host cell AIM2-inflammasome activation after infection of phagocytes with individual clones of an existing library of 312 M. smegmatis (Msme) clones transfected with Mtb-DNA containing cosmids (~40kbp insert size). This library has successfully been used by us during a GoF screen for anti-apoptosis genes in the Mtb genome. The Msme clones with the strongest reduction in IL-1 secretion when compared to wild-type Msme infected cells will be selected and the sequence of the Mtb-DNA insert of the cosmid will be determined. Next, we will use three different strategies to identify the Mtb gene(s) in one of the cosmids mediating the AIM2-inflammasome inhibition. We will make deletion mutants in Mtb which will cover all of the genes of the identified genomic region and analyze the mutants for an expected increase in AIM2-mediated inflammasome activation. Second, in vitro transposon mutagenesis of the cosmid DNA and subsequent DNA sequence analysis will generate an ordered library of transposon mutants. These mutated cosmids will be transfected into Msme and the capacity of reducing AIM2- inflammasome activation will be analyzed. Third, Mtb transposon mutants of the identified genes will be ordered from a repository library (TARGET) and analyzed for increased inflammasome activation. In conclusion, the proposed studies will lead to the identification of Mtb gene(s) important for mediating suppression of host cell AIM2-inflammasome activation. These mutants will enable future in vivo studies in order to determine how important this novel immune evasion strategy is for virulence of Mtb. In addition, these putative virulence genes could be novel targets for drug development and improving recombinant vaccine design.